Design and Synthesis of Aminostilbene-Arylpropenones as Tubulin Polymerization Inhibitors

DOI: 10.1002/cmdc.201402256

Design and Synthesis of Aminostilbene–Arylpropenonesas Tubulin Polymerization InhibitorsAhmed Kamal,*[a, c] G. Bharath Kumar,[a] Sowjanya Polepalli,[b] Anver Basha Shaik,[a]

Vangala Santhosh Reddy,[a] M. Kashi Reddy,[a] Ch. Ratna Reddy,[a] Rasala Mahesh,[a]

Jeevak Sopanrao Kapure,[c] and Nishant Jain[b]

Introduction

Tubulin binding agents are known to play a major role incancer chemotherapy, and well-known tubulin binders such aspaclitaxel, vinca alkaloids, vincristine, and vinblastine are rou-tinely employed as anticancer agents.[1] Microtubule, a polymerof tubulin with a and b subunits, is involved in signal transduc-tion pathways of mammalian cells, particularly in cell division.[2]

Hence, antitubulin agents block the transition from interphaseto mitosis[3] and, thus, effectively block cell-cycle progression,which results in apoptosis.[4] Antimitotic agents derived fromnatural and synthetic products generally exert their effect asmicrotubule stabilizers or polymerizing agents, such as Taxol,paclitaxel, and docetaxel,[5] which block microtubule disassem-bly. They bind at the b-tubulin site in the microtubules and areused in the treatment of carcinomas, such as lung, breast,ovarian, and bladder. In contrast, microtubule destabilizerssuch as colchicinium,[6] vinca alkaloids,[7] and combretastatin A-

4[8] bind at the b-tubulin site in microtubules and cause the de-polymerization of the microtubules. Many of such agents man-ifest different limitations in their clinical utility owing to drugresistance and neurotoxicity;[9] therefore, the development ofnew microtubule targeting agents is of significance.

Combretastatin A-4 (CA-4, 1 a ; Figure 1) is an excellent tubu-lin polymerization inhibitor that binds to the colchicine bind-ing site of tubulin and demonstrates cytotoxicity againsta broad spectrum of human cancer cell lines including multi-drug-resistant cancer cells. However, the in vivo efficiency ofCA-4 is limited owing to its poor pharmacokinetics resultingfrom its high lipophilicity and low water solubility. Structuralmodification of CA-4 has led to the development of a numberof new CA-4 derivatives that are potent tubulin polymerizationinhibitors,[10] including combretastatin A-4 phosphate[11, 12] (CA-4P, 1 b ; Figure 1) and CA-1 disodium phosphate (CA-1P,Oxi 4503) as prodrugs. The water-soluble prodrugs CA-4P andCA-1P have reached the most advanced stage of preclinical de-velopment. Currently, CA-4P is under investigation in phase IItrails for ovarian, lung, and anaplastic thyroid cancers, and itdisplays selective toxicity towards the vasculature of thetumor.[13] However, as its potentiality appears to be uncer-tain,[14] many research groups are looking to improve this scaf-fold by different structural modifications. Some recent studieshave shown that amino-substituted combretastatin derivativeshave potential for further development. In addition, increasedwater solubility of a serine prodrug of combretastatin amine(1 d, ombrabulin, AVE-8062) has a stronger effect on tumorblood flow; this leads to complete blockage of the nutrientsupply to the solid tumor and thereby leads to necrosis.[15, 16]

A series of aminostilbene—arylpropenones were designed andsynthesized by Michael addition and were investigated fortheir cytotoxic activity against various human cancer cell lines.Some of the investigated compounds exhibited significant an-tiproliferative activity against a panel of 60 human cancer celllines of the US National Cancer Institute, with 50 % growth in-hibition (GI50) values in the range from <0.01 to 19.9 mm. Oneof the compounds showed a broad spectrum of antiprolifera-tive efficacy on most of the cell lines, with a GI50 value of<0.01 mm. All of the synthesized compounds displayed cyto-toxicity against A549 (non-small-cell lung cancer), HeLa (cervi-

cal carcinoma), MCF-7 (breast cancer), and HCT116 (colon carci-noma) with 50 % inhibitory concentration (IC50) values rangingfrom 0.011 to 8.56 mm. A cell cycle assay revealed that thesecompounds arrested the G2/M phase of the cell cycle. Twocompounds exhibited strong inhibitory effects on tubulin as-sembly with IC50 values of 0.71 and 0.79 mm. Moreover, dot-blot analysis of cyclin B1 demonstrated that some of the con-geners strongly induced cyclin B1 protein levels. Moleculardocking studies indicated that these compounds occupy thecolchicine binding site of tubulin.

[a] Dr. A. Kamal, G. B. Kumar, A. B. Shaik, V. S. Reddy, M. K. Reddy, C. R. Reddy,R. MaheshMedicinal Chemistry & PharmacologyCSIR-Indian Institute of Chemical Technology Hyderabad, 500 007 (India)E-mail : [email protected]

[b] S. Polepalli, Dr. N. JainCentre for Chemical BiologyCSIR-Indian Institute of Chemical Technology Hyderabad, 500 007 (India)

[c] Dr. A. Kamal, J. S. KapureDepartment of Medicinal ChemistryNational Institute of Pharmaceutical Education & Research (NIPER)Hyderabad-500 037 (India)

Supporting information (1H NMR, 13C NMR, and HRMS spectra of amino-stilbene–arylpropenones 3 a–h, 4 a–h, and 5 a–f) for this article is avail-able on the WWW under http://dx.doi.org/10.1002/cmdc.201402256.

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Structure–activity relationship (SAR) studies of CA-4 analogueshave shown that both hydroxy and amino substituents con-tribute to improve the antimitotic activity and provide the op-portunity to structurally modify this scaffold.[17]

Recently, a series of antiproliferative compounds based onthe (Z)-1-aryl-3-arylamino-2-propen-1-one scaffold of 2 a(Figure 1) were reported to significantly inhibit multidrug-re-sistant cancer cells and were also found to arrest the cells inthe G2/M phase of the cell cycle.[18] A number of tubulin poly-merization inhibitors containing an indole core nucleus havebeen obtained from natural sources and generated throughdesign and synthesis. Some of the indole-containing com-pounds exhibit excellent antimitotic activity such as D-64131,D-24851, BPR0L075, BLF 61-3, and methyl 5-methoxy-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole-2-carboxylate (ATI ) deriva-tives.[19] 6-Methoxy-3-(3’,4’,5’-trimethoxybenzoyl)-1H-indole(BPR0L075, Figure 1), a microtubule depolymerizing agent, ex-hibits antiangiogenic activities.[20, 21] Indibulin {N-(pyridin-4-yl)-[1-(4-chlorbenzyl)indol-3-yl]glyoxyl amide, D-24851; Figure 1 a}is a synthetic small molecule with microtubule destabilizing ac-tivity.[22]

Thus, it is clear from the background presented above thatthe pharmacophores represented by aminostilbene 1 c and ar-

ylpropenones 2 a offer excellentpossibilities for hybridization to-wards the design of improvedtubulin polymerization inhibitors.In addition, the presence of anaryl ring in 2 a provides an op-portunity to introduce a privi-leged indole scaffold in sucha design. We have been previ-ously involved in the develop-ment of new heterocyclic scaf-folds such as combretastatin–amidobenzothiazole conjugates,and benzofurans were shown tobe potential inhibitors of tubulinpolymerization with significantcytotoxic activity.[23, 24] In continu-ation of our earlier efforts andon the basis of literature find-ings, we linked amino-substitut-ed combretastatin with arylami-no-2-propenones to generateaminostilbene–arylpropenonehybrids. The 3,4,5-trimethoxy-phenyl unit constitutes theA ring, which is utilized withoutany structural modification inview of its pharmacophoric im-portance. The aryl units repre-sented by the B ring and C ringare varied to incorporate differ-ent substituents including theindole framework in some cases.

Results and Discussion

Chemistry

The syntheses of aminostilbene–arylpropenones 3 a–h, 4 a–h,and 5 a–f described in this study are outlined in Schemes 1and 2. The final step was performed by Michael condensationbetween (Z)-3-substituted-2-methoxy-5-(3,4,5-trimethoxystyry-l)aniline 12 a/12 b or (Z)-2-amino-6-methoxy-3-(3,4,5-trimethox-ystyryl)phenol (21) and substituted phenylprop-2-yn-1-ones13 a–f or N-substituted-3-indolyl prop-2-yn-1-ones 13 g/13 h inethanol. The key intermediates, 12 a/12 b, were prepared infive steps. 3,4,5-Trimethoxybenzaldehyde (6) was reduced byusing sodium borohydride in methanol to give (3,4,5-trime-thoxyphenyl)methanol (7). This was further treated with phos-phorus tribromide in dichloromethane to produce 5-(bromo-methyl)-1,2,3-trimethoxybenzene (8), which upon further reac-tion with triphenylphosphine in toluene gave 3,4,5-trimethoxy-benzyltriphenylphosphonium bromide (9)[25] in good yield. TheWitting salt thus formed was treated with substituted benzal-dehydes 10 a/10 b in the presence of sodium hydride in di-chloromethane to produce (Z)-1,2,3-trimethoxy-5-(substituted-nitrostyryl)benzenes 11 a/11 b and their E isomers in a 1:1 ratio.

Figure 1. Chemical structure of microtubule targeting agents. CA-4 (1 a), CA-4P (1 b), AVE8063 (1 c), AVE8062 (1 d),(Z)-1-(2-bromo-3,4,5-trimethoxyphenyl)-3-[(3-hydroxy-4-methoxyphenyl)amino]prop-2-en-1-one (2 a), BPR0L075(2 b), D-24851 (Indibulin, 2 c), aminostilbene–arylpropenones 3 a–h, 4 a–h, and 5 a–f.



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Compounds 11 a/11 b were reduced with Zn in acetic acid toafford 12 a/12 b.

Key intermediate 21 was prepared in five sequential steps.Isovaniline (16) was nitrated with nitronium tetrafluoroborateto give 2-nitro 3-hydroxybenzaldehyde (17). This was furthertreated with tert-butyldimethylsilyl chloride (TBDMSCl) and imi-dazole in dichloromethane to produce 3-(tert-butyldimethylsily-loxy)-4-methoxy-2-nitrobenzaldehyde (18). The obtained com-pound was treated with 3,4,5-trimethoxybenzyltriphenylphos-phonium bromide (9) in the presence of sodium hydride in di-chloromethane to produce (Z)-tert-butyl[6-methoxy-2-nitro-3-(3,4,5-trimethoxystyryl)phenoxy]dimethylsilane (19). Then, 19

was further reduced with Zn in acetic acid to give (Z)-2-(tert-butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)ani-line (20). Later, 20 was deprotected by using tetrabutylammo-nium fluoride (TBAF, 1 n in THF) to give derivative 21.[25]

Aryl/heteroaryl prop-2-yn-1-ones 13 a–g were prepared intwo steps, as shown in Scheme 3. Substituted benzaldehydes22 a–g were treated with ethynylmagnesium bromide (0.5 n inTHF) to give aryl/heteroaryl prop-2-yn-1-ols 23 a–g. These sec-ondary alcohols were oxidized with 2-iodoxybenzoic acid (IBX)in dimethyl sulfoxide (DMSO) to produce aryl/heteroaryl prop-2-yn-1-ones 13 a–g in good yields.

Scheme 1. Reagents and conditions : a) NaBH4, MeOH, 3 h, 15–20 8C; b) PBr3, CH2Cl2, 2 h, 15–20 8C; c) PPh3, toluene, 12 h, 80 8C; d) NaH, CH2Cl2, 18 h, 15–20 8C;e) Zn AcOH, 4 h, RT; f) EtOH, RT, 4 h; g) TBAF (1 m in THF), THF 0 8C, 4 h; h) Zn, ammonium formate, EtOH, 4 h; i) TFA, CH2Cl2, 6 h, 0–5 8C.




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Final compounds 3 e, 4 e, and 5 e were prepared by the re-duction of 3 d, 4 d, and 5 d, respectively, with Zn and ammoni-um formate in methanol. Compounds 14 a, 14 b, and 14 cupon deprotection with TBAF (1 n in THF) afforded 3 c, 4 c, and5 c, respectively. Compounds 3 g and 4 g were prepared from15 a and 15 b by deprotection of the tert-butoxycarbonyl (Boc)group by using trifluoroacetic acid (TFA) in dichloromethane.

Biological studies

Antiproliferative activity

Congeners 3 a–h, 4 a–h, and 5 a–f (Scheme 1) and 2 were evaluat-ed for their cytotoxic activity bythe US National Cancer Institute(NCI) in a 60-cell-line screen.Among the 22 compounds, com-pounds 3 g, 4 a, 4 f, 4 g, and 4 hwere selected for a preliminarytest at a single concentration(10 mm), and these compoundsexhibited significant growth in-hibition. Apart from 4 h, theother compounds were furtherevaluated in a five-dose screen.Compounds 3 g, 4 a, 4 f, and 4 gshowed remarkable antiprolifera-tive activity in a nine cancerpanel of this NCI screen (leuke-mia, lung, colon, central nervoussystem, melanoma, ovary, kidney,prostate, and breast cancers).Compound 3 g with a methoxy

group in the B ring and an indole moiety in the C ringemerged as the most promising in this series. Notably, 3 gdemonstrated a 50 % growth inhibition (GI50) value of<0.01 mm in most of the cell lines of the 60-cell-line panel;thus, it possesses a broad spectrum of cytotoxicity. Com-pounds 4 a, 4 f, and 4 g have a common dimethoxy group inthe B ring as a substituent, and presumably, the presence oftrimethoxy (for 4 a), 4-trifluoromethoxy (for 4 f), and indole (for4 g) groups in the C rings of these molecules resulted in cyto-toxic activity in the NCI screen (see Table 1). Therefore, all ofthe compounds of this series were evaluated for their antiproli-ferative activities. Interestingly, similar to the NCI screening re-sults, compounds 3 a–h (Figure 2) possessing a methoxy groupin the B ring showed profound cytotoxic activity with a 50 %inhibitory concentration (IC50) value range of 0.011–4.68 mm.Among them, compound 3 e, which possess meta-amine andpara-methoxy groups on the C ring, inhibited the growth ofA549 cells with a IC50 value of 11 nm, as shown in Table 2.Moreover, compounds 4 a–h with meta,para-dimethoxy-substi-tuted B rings also inhibited cell growth with an IC50 value inthe range from 0.09 to 7.59 mm. Notably, 4 e with meta-aminoand para-methoxy substitutions in the C ring inhibited growthof A549 cells with an IC50 value of 90 nm. In addition, hydroxyand methoxy substituents in the B rings of 5 a–h resulted inless-potent cytotoxic activities with IC50 values of 0.91–7.33 mm.Compound 5 e with meta-amino and para-methoxy groups inthe C ring showed potent cytotoxic activity against MCF-7 cellswith an IC50 value of 0.91 mm. Taken together, these results sug-gest that congeners with methoxy or dimethoxy moieties inthe B ring and indole or meta-amino and para-methoxy substi-tutions in the C ring exhibit potent antiproliferative activities.Moreover, on the basis of the five-dose results of the NCI, we

Scheme 2. Reagents and conditions : a) Nitronium tetrafluoroborate, CH3NO2/CH2Cl2 (6:4), �40 8C, 8 h; b) TBDMSCl,imidazole, CH2Cl2, 3 h, 15–20 8C; c) NaH, CH2Cl2, 18 h, 15–20 8C; d) Zn AcOH, 4 h, RT; e) EtOH, RT, 4 h; f) TBAF (1 m

in THF), THF 0 8C, 4 h; g) Zn, ammonium formate, EtOH, 4 h.

Scheme 3. Reagents and conditions : a) Ethynylmagnesium bromide (0.5 n inTHF), THF, 8 h, 15–20 8C; b) IBX, DMSO, 15–20 8C, 2 h.




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hypothesize that these compounds inhibit a common target inall cell lines.

Dot-blot analysis for cyclin B1

Given that the congeners contain a combretastatin moiety,these molecules are expected to interfere with tubulin poly-

merization and arrest cells at the G2/M phase of the cellcycle.[26] As the majority of the molecules in this series exhibitsignificant cytotoxicity, we tested their ability to induce cy-clin B1 protein, a marker for mitosis. To test this possibility,A549 cells were treated with various congeners at 1 mm con-centrations for 24 h. Subsequently, dot-blot analysis for cy-clin B1 indicated that most of the congeners strongly inducedcyclin B1 protein levels. Notably, 3 e, 3 g, 4 e, and 5 e robustlyactivated cyclin B1. These results suggest that the congenersindeed arrest cells at the G2/M phase of the cell cycle; actinwas employed as a loading control (Figure 3).

Effect on cell-cycle arrest

To investigate the effect of potential compounds 3 e, 3 g, and4 e on the cell-cycle distribution of A549 cells, flow cytometryanalysis was performed at a concentration of 1 mm for 24 h.

Table 1. In vitro cytotoxic effect of conjugates 3 g, 4 a, 4 f, 4 g, and 1 a against a panel of 60 human cancer cells.

Cell line GI50 [mm][a] Cell line GI50 [mm][a]

3 g[b] 4 a[c] 4 f[d] 4 g[e] 1 a[f] 3 g[b] 4 a[c] 4 f[d] 4 g[e] 1 a[f]

Leukemia OvarianCCRF-CEM <0.01 2.93 3.55 1.37 0.002 IGROV1 <0.01 6.06 7.07 1.23 0.015HL60(TB) <0.01 3.24 2.80 –[h] –[h] OVCAR-3 <0.01 2.31 3.48 1.72 0.001K-562 <0.01 3.38 2.93 1.54 0.002 OVCAR-4 <0.01 2.91 7.54 1.52 0.015MOLT-4 <0.01 4.62 3.99 2.53 0.003 OVCAR-5 <0.01 3.64 8.20 2.45 0.1RPMI-8226 <0.01 3.04 3.66 0.63 0.003 OVCAR-8 <0.01 4.70 4.59 3.33 0.003SR <0.01 2.03 2.75 0.46 0.003 NCI/ADR-RES <0.01 3.12 3.04 3.49 0.001

SK-OV-3 <0.01 2.91 5.38 12.3 0.063

Non-small-cell lung RenalA549/ATCC <0.01 5.14 4.54 14.5 0.015 786-0 <0.01 3.76 4.34 2.04 0.1HOP-62 <0.01 2.46 7.85 11.6 0.002 A498 <0.01 2.40 1.83 1.32 0.006HOP-92 0.01 1.14 12.6 1.50 0.002 ACHN <0.01 3.99 5.56 1.75 0.006NCI-H226 0.02 5.45 19.9 7.28 0.003 CAKI-1 <0.01 3.37 4.96 1.58 0.025NCI-H23 <0.01 4.22 4.38 3.75 0.003 RXF 393 –[h] –[h] 2.18 1.69 0.002NCIH322M –[g] 7.71 1.10 1.20 0.003 SN12C 0.02 4.03 4.85 2.44 0.006NCI-H460 <0.01 3.93 3.34 5.60 0.007 TK-10 –[g] 5.93 4.79 1.86 0.1NCI-H522 –[h] –[h] 1.96 1.76 0.001 UO-31 <0.01 2.63 5.42 0.32 0.019

Colon ProstateCOLO-205 <0.01 2.31 2.98 1.51 0.1 PC-3 <0.01 2.89 –[h] 3.44 0.001HCC-2998 <0.01 8.92 3.71 3.34 0.063 DU-145 <0.01 3.64 4.11 2.06 0.001HCT-116 <0.01 2.78 3.58 1.76 0.003HCT-15 <0.01 3.08 3.42 2.32 0.003 BreastHT29 <0.01 3.12 3.07 2.28 0.1 MCF7 <0.01 2.34 4.08 0.72 0.005KM12 <0.01 3.21 3.69 3.69 0.005 MDA-MB-31/ATCC <0.01 4.01 3.00 1.73 0.001SW-620 <0.01 4.12 4.09 1.63 0.003 HS 578T <0.01 2.76 6.44 2.21 0.001

BT-549 <0.01 –[h] 5.90 1.64 10Melanoma T-47D –[g] 1.68 4.16 1.55 10LOX IMVI <0.01 3.54 5.95 0.44 0.003 MDA-MB-435 <0.01 1.88 3.16 1.28 –[g]

MALME-3M 5.00 2.91 14.7 1.27 0.019 MDA-MB-435 <0.01 1.88 3.16 1.28 –[g]

M14 <0.01 2.53 3.46 2.87 0.002MDA-MB-435 <0.01 1.22 1.84 2.43 0.001 CNSSK-MEL-2 <0.01 4.54 –[h] 2.21 0.003 SF-268 <0.01 6.04 8.90 3.57 0.006SK-MEL-28 1.59 4.28 10.4 1.86 0.007 SF-295 <0.01 2.40 4.12 7.94 0.003SK-MEL-5 <0.01 1.69 3.73 3.14 0.003 SF-539 <0.01 1.92 2.26 1.70 0.002UACC-257 –[g] –[g] 4.33 3.16 0.003 SNB-19 <0.01 4.49 4.97 3.92 0.003UACC-62 4.38 3.11 6.86 1.39 0.005 SNB-75 <0.01 1.39 2.65 1.47 0.007

U251 <0.01 3.84 3.46 3.84 0.007

[a] Data are reported as the GI50 value (concentration required to cause 50 % inhibition of cell growth after an incubation time of 48 h). [b] NSC 777174.[c] NSC 777180. [d] NSC 773189. [e] NSC 777183. [f] NSC 613729. [g] Not active. [h] Not tested.

Figure 2. Structure–activity relationship of aminostilbene–arylpropenones.




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The results indicate that they caused G2/M cell-cycle arrestwith a large accumulation of cells. Cells treated with 3 e at1 mm showed 76.4 % arrest of cells in the G2/M phase, whereasA549 cells treated with 3 g and 4 e at 1 mm showed arrest ofcells in the G2/M phase by 73 and 71 %, respectively (Figure 4).

In contrast, vehicle- or DMSO-treated cells showed the majorityof the population in the G1 phase (67.7 %) of the cell cycle.[27]

Inhibition of tubulin polymerization

To further investigate whether the antiproliferative activities ofcompounds 3 e, 3 g, and 4 e were derived from interactionwith tubulin, they were evaluated for their inhibition of tubulinpolymerization (Table 3). The tubulin assembly assays revealedthat these molecules showed potent inhibition of tubulin poly-merization with IC50 values of 0.71 mm for 3 e and 0.79 mm for3 g ; these values are comparable to that for CA-4 (1 a, Table 3).

Effect of 3 e, 3 g, and 4 e on microtubule network

The presence of anomalous spindle fibers owing to altered mi-crotubule dynamics is a hallmark of cells treated with microtu-bule inhibitors.[28] These aminostilbene–arylpropenones signifi-cantly decreased cell growth, inhibited tubulin assembly, andstalled cells in the G2/M phase of the cell cycle. Therefore, wewere prompted to evaluate their ability to disrupt microtubulenetworks in the cells.[29] Consequently, A549 cells were treatedwith 3 e, 3 g, and 4 e at 1 mm concentrations for 24 h. Immuno-fluorescence analysis revealed that the cells exhibited roundedmorphology typical of a mitotic-arrest population, and more-over, chromatin was condensed in the nuclei, which suggestsmetaphase arrest (Figure 5).

Effect of 3 e, 3 g, and 4 e on cytosolic tubulin

Given that the aminostilbene–arylpropenones markedly inhibit-ed in vitro tubulin polymerization, we investigated their abilityto inhibit endogenous tubulin assembly. Microtubules exist ina dynamic equilibrium between free and polymerized tubu-lin,[30] and the amount of free tubulin increases in cells chal-lenged with microtubule inhibitors. To test this possibility,A549 cells were treated with 3 e, 3 g, and 4 e at 1 mm concen-trations for 24 h. Subsequently, the cells were permeabilizedwith detergents, and soluble (free tubulin) and polymerizedfractions were collected. Immunoblot analysis suggested thatthe DMSO-treated cells contain fairly equal amounts of tubulinin both fractions (Figure 6.). In comparison, the compound-treated cells showed a significant amount of tubulin in thesoluble fraction. Thus, these results suggest that the congenersstrongly inhibit tubulin assembly in in vitro assays as well as incell-based assays.

Molecular modeling studies

A molecular modeling study was performed to elucidate thebinding characters of conjugates 3 e, 3 g, and 4 e with tubulin.Coordinates of the protein structure of tubulin–colchicine wasobtained from the Protein Data Bank (PDB ID 3E22).[32] Dockingstudies of all compounds were accomplished into the colchi-cine binding site of tubulin by using AutoDock 4.2 software.[ 33]

Congeners 3 a–h, 4 a–h, and 5 a–f possess two cis-olefinicunits, and they were expected to occupy the colchicine bind-

Table 2. In vitro cytotoxic effect of aminostilbene–arylpropenones 3 a–h,4 a–h, and 5 a–f.

Compd IC50 [mm][a]

A549[b] HeLa[c] MCF-7[d] HCT116[e]

3 a 1.68�0.12 0.26�0.03 0.27�0.03 1.51�0.023 b 3.51�0.3 0.25�0.02 2.95�0.78 3.98�0.83 c 0.77�0.04 0.23�0.012 1.59�0.2 0.83�0.053 d 3.81�0.51 2.21�0.09 4.68�0.47 1.29�0.143 e 0.011�0.001 0.052�0.013 0.025�0.001 0.028�0.0043 f 2.69�0.23 0.26�0.024 1.32�0.09 2.95�0.33 g 0.015�0.003 0.053�0.001 0.031�0.001 0.033�0.0013 h 0.67�0.04 0.46�0.02 1.07�0.049 0.37�0.054 a 6.25�0.24 3.72�0.35 3.08�0.1 4.32�0.0214 b 0.28�0.02 1.58�0.07 5.67�0.27 0.3�0.014 c 0.92�0.01 0.91�0.016 2.09�0.04 3.24�0.134 d 4.15�0.09 1.2�0.08 1.55�0.31 4.79�0.254 e 0.09�0.001 0.19�0.045 0.11�0.08 0.19�0.084 f 5.21�0.019 2.86�0.06 4.75�0.07 7.59�0.494 g 8.56�0.02 1.67�0.05 3.02�0.05 2.48�0.214 h 0.14�0.015 0.89�0.03 3.39�0.2 1.95�0.65 a 2.25�0.024 1.56�0.014 2.23�0.15 3.63�0.415 b 7.08�0.56 2.78�0.38 3.19�0.4 2.69�0.325 c 1.58�0.09 4.17�0.14 2.2�0.16 6.76�0.545 d 7.33�0.21 2.66�0.42 2.6�0.02 3.98�0.735 e 1.31�0.02 2.51�0.08 0.91�0.04 1.75�0.0265 f 1.31�0.4 7.08�0.042 2.12�0.063 1.94�0.021 a (CA-4) 0.031�0.001 0.058�0.007 0.058�0.003 0.398�0.46

[a] Concentration required to inhibit 50 % cell growth following 48 htreatment with the tested drug. Values represent mean� standard devia-tion from three different experiments performed in triplicate. [b] A549:non-small cell lung cancer. [c] HeLa: cervix cancer. [d] MCF-7: breastcancer. [e] HCT116: colon cancer cell line.

Figure 3. Dot-blot analysis of cyclin B1: A549 cells were treated with 1 mm

concentrations of aminostilbene–arylpropenones for 24 h. Later, cells wereharvested and whole-cell lysates were blotted on nitrocellulose membranes.The blotted proteins were probed with antibodies against cyclin B1. Actinwas employed as loading control.




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ing site of tubulin. Conjugate 3 e exhibited a hydrogen-bond-ing interaction between the oxygen atom of the carbonylgroup on the methoxyaniline ring with bLys254. Hydrophobicinteractions were found between the methoxyaniline ring of3 e with aAsn101 and bAsn249. The amine group of methoxya-niline was involved in a hydrogen-bonding interaction withaAla250 and in a hydrophobic interaction with bAsn250. TheA ring (trimethoxyphenyl) of 3 e was buried in the hydrophobicpockets of bLys254, bAsn258, bVal238, bLeu242, bAla250, andbLeu255 in a manner similar to that of the trimethoxyphenylgroup of colchicine. Some hydrophobic interactions wereformed by the B ring in both the a- and b-tubulin interfacewith aSer178, aThr179, bLys352, and bAsn258. Conjugate 3 gshowed a hydrogen-bonding interaction between indole andaGlu183, and this indole ring was buried in hydrophobic inter-actions with aTyr224, aGly142, and aAla180.

The trimethoxyphenyl group (A ring) showed hydrophobicinteractions with bLeu255, bLeu248, bAla250, bLys254, andbLys255, and the methoxy group of the A ring established a hy-drogen-bonding interaction with bLeu255. The methoxy group

of the B ring showed hydropho-bic interactions with bLeu254,bLeu255, and bAla354. The car-bonyl group on the indole ringestablished a hydrophobic inter-action with bLeu248. Conjugate4 e established a hydrogen-bonding interaction betweenthe methoxy group on anilineand bLys254. The carbonylgroup on the methoxyanilinemoiety showed a hydrogen-bonding interaction withaThr179 and a hydrophobic in-teraction with aSer178. The me-thoxyaniline group showed hy-drophobic interactions withaTyr224, bGly247, and aGln11.The trimethoxyphenyl group(A ring) was buried in the hydro-phobic pockets of bLeu255,bLys254, bLeu248, and bAla250.

The dimethoxyphenyl group (B ring) showed hydrophobic in-teractions with bMet259, aAla180, bAla316, bLeu255, andbLys352. Conjugates 3 e, 3 g, and 4 e all interacted with the

Figure 4. Antimitotic effects of 3 e, 3 g, and 4 e by fluorescence-activated cell sorting (FACS) analysis. Induction ofcell-cycle G2/M arrest by compounds 3 e, 3 g, and 4 e. A549 cells were harvested after treatment at 1 mm for 24 h.Untreated cells and DMSO-treated cells served as controls. The percentage of cells in each phase of the cell cyclewas quantified by flow cytometry.

Table 3. Antitubulin activity of aminostilbene–arylpropenones 3 e, 3 g,and 4 e.[a]

Compd IC50 [mm]

3 e 0.71�0.013 g 0.79�0.044 e 1.68�0.431 a (CA-4) 0.91�0.12

[a] Inhibition of tubulin polymerization (IC50) values for 3 e, 3 g, and 4 ewere determined from tubulin polymerization assays.

Figure 5. Effect of 3 e, 3 g, and 4 e on microtubules and nuclear condensa-tion. A549 cells were independently treated with 3 e, 3 g, and 4 e at 1 mm for24 h. Following treatment, cells were fixed and stained for tubulin by using4’,6-diamidino-2-phenylindole (DAPI) as a counterstain. Photographs weretaken by using an Olympus confocal fluorescence microscope equippedwith fluorescein isothiocyanate (FITC) and DAPI filter settings. Cells stainedfor tubulin and DAPI from the same field of views are represented.




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a,b-tubulin interface in the colchicine binding pocket(Figure 7).

Conclusions

A library of novel aminostilbene–arylpropenones (i.e. , 3 a–h,4 a–h, and 5 a–f) comprising an A,B,C ring system was synthe-

sized by using the Michael addition reaction, and the com-pounds were evaluated for their cytotoxic activity againsthuman cancer cell lines. Representative congeners 3 g, 4 a, 4 f,and 4 g were evaluated in a 60-cell-line panel of the NCI five-dose screen. Among them, 3 g, which possesses a para-me-thoxy group in the B ring and an indole moiety as the C ring,exhibited excellent cytotoxicity in most human cancer celllines with GI50 values <0.01 mm. Compounds 3 a–h, 4 a–h, and5 a–f all displayed antiproliferative activity against four humancancer cell lines, including A549 (non-small cell lung cancer),HeLa (cervical carcinoma), MCF-7 (breast cancer), and HCT116(colon carcinoma) with IC50 values ranging from 0.011 to8.56 mm, and some of these values are comparable to that ofCA-4 (1 a) The cell-cycle assays revealed that congeners 3 e,3 g, and 4 e caused cell-cycle arrest and accumulated cells inthe G2/M phase. Furthermore, these compounds effectively in-hibited microtubule assembly and induced cyclin B1 proteinexpression. As hypothesized, lead compounds 3 e, 3 g, and 4 eexert their cytotoxic activity by inhibition of tubulin polymeri-

Figure 6. Distribution of tubulin in polymerized versus soluble fractions ana-lyzed by immunoblotting in A549 cells treated with 3 e, 3 g, and 4 e. A549cells were treated with 1 mm of 3 e, 3 g, and 4 e for 24 h. The fractions con-taining soluble and polymerized tubulin were collected and separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis. Tubulin was de-tected by immunoblot analysis. S = soluble, P = polymerized.

Figure 7. Interaction of 3 e, 3 g, 4 e, and 1 a (CA-4) with the colchicine binding site of tubulin. Probable hydrogen bonds are shown in red. This figure wasgenerated by using the PyMOL software.




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zation, with IC50 values of 0.71, 0.79, and 1.68 mm, respectively.The binding of these conjugates is at the colchicine bindingsite of tubulin, as indicated by molecular modeling studies.These results demonstrate that aminostilbene–arylpropenonesare potent inhibitors of tubulin polymerization, and they arealso amenable for further structural modifications in the dis-covery and development of effective chemotherapeutics forthe treatment of cancer.

Experimental Section

Chemistry

General : All chemicals and reagents were obtained from Sigma–Al-drich (St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson MattheyCompany, Ward Hill, MA, USA), or Spectrochem Pvt. , Ltd. (Mumbai,India), and were used without further purification. Reactions weremonitored by TLC performed on glass plates coated with silica gelcontaining 60 GF254, and visualization was performed by UV lightor iodine indicator. Column chromatography was performed withMerck 60–120 mesh silica gel. 1H NMR spectra were recorded witha Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR-Unity(400 MHz) instrument. Chemical shifts (d) are reported in ppmdownfield from internal (CH3)4Si standard. ESI-MS data were record-ed with a Micromass Quattro LC instrument by using ESI + soft-ware with a capillary voltage of 3.98 kV and an ESI mode positiveion trap detector. HRMS data were recorded with a QSTAR XLHybrid MS–MS mass spectrometer. Melting points were deter-mined with an Electrothermal melting point apparatus.

(Z)-1,2,3-Trimethoxy-5-(4-methoxy-3-nitrostyryl)benzene (11 a): Asolution of 4-methoxy-3-nitrobenzaldehyde (10 a ; 2.0 g, 0.011 mol)and triphenyl(3,4,5-trimethoxybenzyl)phosphonium bromide (9 ;6.34 g, 0.012 mol) in dry CH2Cl2 (100 mL) was stirred under a nitro-gen atmosphere. Sodium hydride (1.06 g, 0.044 mol) was added tothe mixture at 0 8C. Then, the temperature of the mixture wasslowly increased to RT, and the mixture was stirred for 18 h. Theprogress of the reaction was monitored by TLC (EtOAc/hexane =3:7), and water was added after completion of reaction (until foam-ing stopped). The organic layer was separated, and the aqueouslayer was extracted with CHCl3. The organic layer was washed withbrine, dried with anhydrous Na2SO4, and concentrated under re-duced pressure to afford the crude compound, which was purifiedby column chromatography (15 % EtOAc/hexanes). Yield: 1.8 g,42 %; 1H NMR (300 MHz, CDCl3): d= 7.79 (d, J = 2.26 Hz, 1 H), 7.42(dd, J = 2.26 Hz, 8.3 Hz, 1 H), 6.94 (d, J = 8.3 Hz, 1 H), 6.58 (d, J =12.09 Hz, 1 H), 6.47 (s, 2 H), 6.46 (d, J = 12.09 Hz, 1 H), 3.93 (s, 3 H),3.85 (s, 3 H), 3.71 ppm (s, 6 H); MS (ESI): m/z : 346 [M+H]+ .

(Z)-5-(3,4-Dimethoxy-5-nitrostyryl)-1,2,3-trimethoxybenzene(11 b): Compound 11 b was prepared according to the method de-scribed for compound 11 a by employing 4,5-dimethoxy 3-nitro-benzaldehyde (10 b ; 2.0 g, 9.5 mmol), triphenyl(3,4,5-trimethoxy-benzyl)phosphonium bromide (9 ; 5.45 g, 0.0104 mol), and sodiumhydride (0.910 g, 0.038 mol) to obtain pure 11 b as a yellow solid.Yield: 1.5 g, 42 %; 1H NMR (300 MHz, CDCl3): d= 7.21 (s, 1 H), 6.93 (s,1 H), 6.56 (d, J = 11.8 Hz, 1 H), 6.43 (s, 2 H), 6.40 (d, J = 11.8 Hz, 1 H),3.91 (s, 3 H), 3.79 (s, 3 H), 3.71 (s, 6 H), 3.66 ppm (s, 3 H); MS (ESI): m/z : 376 [M+H]+ .

(Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)aniline (12 a): Zinc(1.46 g, 0.023 mol) was added to a stirred solution of 11 a (2.0 g,5.78 mmol) in acetic acid (20 mL) under a nitrogen atmosphere at0 8C. Then, the temperature of the mixture was slowly increased to

RT, and the mixture was stirred for 6 h. The progress of the reac-tion was monitored by TLC (EtOAc/hexane = 1:1). The mixture wasbasified with saturated NaHCO3 solution and then extracted withEtOAc. The organic layer was separated and washed with brine,dried with anhydrous Na2SO4, and concentrated under reducedpressure to afford the crude compound, which was purified bycolumn chromatography (25 % EtOAc/hexanes). Yield: 1.4 g, 77 %;1H NMR (300 MHz, CDCl3): d= 6.91 (d, J = 2.26 Hz, 1 H), 6.80 (dd, J =2.26 Hz, 8.31 Hz, 1 H), 6.73 (d, J = 8.31 Hz, 1 H), 6.42–6.36 (m, 2 H),6.33 (dd, J = 1.51 Hz, 9.82 Hz, 2 H), 3.86 (s, 3 H), 3.81 (s, 6 H),3.65 ppm (s, 3 H); MS (ESI): m/z : 316 [M+H]+ .

(Z)-2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)aniline (12 b): Com-pound 12 b was prepared according to the method described forcompound 12 a by employing 11 b (2.0 g, 5.32 mmol) and zinc(1.34 g, 0.0213 mol) to obtain pure 12 b as a yellow solid. Yield:1.5 g, 82 %; 1H NMR (300 MHz, CDCl3): d= 6.70 (d, J = 1.51 Hz, 1 H),6.68 (s, 2 H), 6.55 (s, 1 H), 6.45 (d, J = 12.1 Hz, 1 H), 6.36 (d, J =12.1 Hz, 1 H), 3.85 (s, 3 H), 3.83 (s, 3 H), 3.70 ppm (s, 3 H); MS (ESI):m/z : 346 [M+H]+ .

(Z)-3-({2-Methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3 a): 1-(3,4,5-Trimethox-yphenyl)prop-2-yn-1-one (13 a ; 69.8 mg, 0.317 mmol) was added toa stirred solution of 12 a (100 mg, 0.317 mmol) in ethanol (5 mL).The mixture was stirred at 25–35 8C for 4 h, and the progress ofthe reaction was monitored by TLC (hexane/EtOAc = 6:4). Then,water was added to the mixture. A yellow color solid appeared,which was filtered and washed with ethanol. Yield: 120 mg, 71 %;m.p. 61–63 8C; 1H NMR (500 MHz, CDCl3): d= 12.09 (d, J = 12.5 Hz,1 H), 7.23–7.21 (m, 1 H), 7.20 (s, 2 H), 7.15 (d, J = 1.8 Hz, 1 H), 6.96(dd, J = 6.56, 1.8 Hz, 1 H), 6.82 (d, J = 8.3 Hz, 1 H),6.54 (s, 2 H), 6.49(q, J = 12.2 Hz, 2.44 Hz, 2 H), 5.92 (d, J = 7.9 Hz, 1 H), 3.96 (s, 3 H),3.93 (s, 6 H), 3.90 (s, 3 H), 3.85 (s, 3 H), 3.71 ppm (s, 6 H); 13C NMR(75 MHz, CDCl3): d= 189.7, 152.9, 152.8, 147.5, 142.9, 141.0, 137.0,134.6, 132.7, 129.9, 129.2, 129.1, 124.6, 113.3, 110.6, 105.7, 104.5,94.1, 60.8, 56.16, 55.98, 55.9 ppm; IR (KBr): nmax = 3452, 2934, 2859,1630, 1588, 1553, 1505, 1472, 1429, 1408, 1363, 1326, 1275, 1199,1166, 1125, 1002, 914, 845, 769, 580 cm�1; MS (ESI): m/z : 536[M+H]+ ; HRMS (ESI): m/z : calcd for C30H34O8N: 536.22789 [M+H]+ ;found: 536.22690.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-({2-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (3 b): Com-pound 3 b was prepared according to the method described forcompound 3 a by employing 1-(2-bromo-3,4,5-trimethoxyphenyl)-prop-2-yn-1-one (13 b ; 94.9 mg, 0.317 mmol) to obtain pure 3 b asa yellow solid. Yield: 150 mg, 77 %; m.p. 54–57 8C; 1H NMR(300 MHz, CDCl3): d= 11.92 (d, J = 12.8 Hz, 1 H), 7.28–7.19 (m, 1 H),7.14 (s, 1 H), 6.97 (d, J = 8.3 Hz, 1 H), 6.87 (s, 1 H), 6.81 (d, J = 8.3 Hz,1 H), 6.49 (d, J = 9.06 Hz,4 H), 5.64 (d, J = 8.30 Hz, 1 H), 3.94 (s, 3 H),3.91 (s, 3 H), 3.90 (s, 3 H), 3.87 (s, 3 H), 3.84 (s, 3 H), 3.70 ppm (s, 6 H);13C NMR (75 MHz, CDCl3): d= 192.2, 152.9, 152.6, 147.5, 144.0,142.8, 138.3, 137.13, 132.6, 130.01, 129.4, 129.1, 125.9, 124.8, 113.6,110.6, 107.9, 106.1, 105.7, 98.4, 61.09, 60.91, 60.91, 56.08, 55.99,55.91 ppm; IR (KBr): nmax = 3418, 2933, 2839, 2359, 1629, 1581,1501, 1465, 1424, 1328, 1379, 1202, 1163, 1125, 1102, 1007, 928,850, 793, 743 cm�1; MS (ESI): m/z : 614 [M+H]+ ; HRMS (ESI): m/z :calcd for C30H33O8NBr: 614.13841 [M+H]+ ; found: 614.13838.

(Z)-1-[3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl]-3-({2-me-thoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (14 a): Compound 14 a was prepared according to the methoddescribed for compound 3 a by employing 1-[3-(tert-butyldimethyl-silyloxy)4-methoxyphenyl]prop-2-yn-1-one (13 c ; 92.07 mg,




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0.317 mmol) to obtain pure 14 a as a yellow solid. Yield: 150 mg,78 %.

(Z)-1-(3-Hydroxy-4-methoxyphenyl)-3-({2-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (3 c): 1 m TBAF inTHF (0.3 mL, 0.272 mmol) was added to a stirred solution of 14 a(150 mg, 0.247 mmol) in THF (15 mL) at 10–15 8C. Then, the tem-perature of the mixture was slowly increased to RT, and the mix-ture was stirred for 6 h. The progress of the reaction was moni-tored by TLC (hexane/EtOAc = 1:1). Upon completion of the reac-tion, THF was evaporated, and the mixture was partitioned be-tween water and EtOAc. The compound was purified by columnchromatography (hexane/EtOAc = 6:4). Yield: 100 mg, 82 %; m.p.70–72 8C; 1H NMR (300 MHz, CDCl3): d= 12.06 (d, J = 12.1 Hz, 1 H),7.50 (d, J = 6.7 Hz, 2 H), 7.24–7.18 (m, 1 H), 7.12 (s, 1 H), 6,96–6.87(m, 2 H), 6.79 (d, J = 8.3 Hz, 1 H), 6.49 (d, J = 13.6 Hz, 4 H), 5.91 (d,J = 8.3 Hz, 1 H), 5.68 (s, 1 H), 3.94 (s, 6 H), 3.85 (s, 3 H), 3.71 ppm (s,6 H); 13C NMR (75 MHz, CDCl3): d= 187.5, 152.9, 147.3, 140.3, 137.4,133.8, 132.2, 130.02, 129.8, 129.5, 128.9, 126.3, 123.4, 122.7, 122.4,121.7, 112.7,110.4, 109.4, 105.79, 96.4, 60.9, 55.93 ppm; IR (KBr):nmax = 3431, 2992, 2945, 2843, 1628, 1614, 1581, 1533, 1501, 1475,1439, 1412, 1348, 1323, 1272, 1241, 1151, 1124, 1081, 1045, 1008,871 cm�1; MS (ESI): m/z : 492 [M+H]+ ; HRMS (ESI): m/z : calcd forC28H30O7N: 492.20168 [M+H]+ ; found: 492.20145.

(Z)-1-(4-Methoxy-3-nitrophenyl)-3-({2-methoxy-5-[(Z)-3,4,5-trime-thoxystyryl]phenyl}amino)prop-2-en-1-one (3 d): Compound 3 dwas prepared according to the method described for compound3 a by employing 1-(4-methoxy-3-nitrophenyl)prop-2-yn-1-one(13 d ; 65.05 mg, 0.317 mmol) to obtain pure 3 d as a yellow solid.Yield: 130 mg, 79 %; m.p. 164–166 8C; 1H NMR (300 MHz, CDCl3):d= 12.15 (d, J = 12.8 Hz, 1 H), 8.44 (s, 1 H), 8.14 (d, J = 9.06 Hz, 1 H),7.32–7.26 (m, 1 H), 7.12 (d, J = 8.13 Hz, 2 H), 6.98 (d, J = 8.3 Hz, 1 H),6.81 (d, J = 9.06 Hz, 1 H), 6.50 (d, J = 8.3 Hz, 4 H), 5.91 (d, J = 7.55 Hz,1 H), 4.02 (s, 3 H), 3.96 (s, 3 H), 3.85 (s, 3 H), 3.70 ppm (s, 6 H);13C NMR (75 MHz, CDCl3): d= 186.9, 155.0, 152.9, 147.6, 143.8,139.2, 137.15, 133.0, 132.6, 131.6, 130.0, 129.4, 129.07, 128.9, 125.0,124.9, 113.7, 113.0, 110.7, 105.7, 93.2, 60.9, 55.9,56.7, 56.03 ppm; IR(KBr): nmax = 3427, 2934, 2837, 1630, 1612, 1579, 1531, 1503, 1473,1437, 1414, 1350, 1325, 1270, 1237, 1149, 1183, 1125, 1085, 1047,1012, 879, 682 cm�1; MS (ESI): m/z : 521 [M+H]+ ; HRMS (ESI): m/z :calcd for C28H29O8N2 : 521.19184 [M+H]+ ; found: 521.19148.

(Z)-1-(4-Amino-3-methoxyphenyl)-3-({2-methoxy-5-[(Z)-3,4,5-tri-methoxystyryl]phenyl}amino)prop-2-en-1-one (3 e): Zinc(77.12 mg, 1.22 mmol) and ammonium formate (78.34 mg,1.22 mmol) were added to a stirred solution of 3 d (150 mg,0.306 mmol) in ethanol (10 mL) at RT. The mixture was stirred for4 h, and the progress of the reaction was monitored by TLC(EtOAc/hexane = 7:3). The mixture was then filtered through a bedof Celite, and the filtrate was evaporated under reduced pressure.Then, water (10 mL) and EtOAc (30 mL) were added to the concen-trate. The organic layer was separated, dried with anhydrousNa2SO4, and concentrated to afford the crude product, which wasfurther purified by column chromatography (EtOAc/hexane = 6:4)to obtain a pure yellow solid. Yield: 110 mg, 78 %; m.p. 71–73 8C;1H NMR (300 MHz, CDCl3): d= 12.04 (d, J = 12.04 Hz, 1 H), 7.36–7.32(m, 2 H), 7.21–7.13 (m, 1 H), 7.12 (d, J = 1.51 Hz, 1 H), 6.95–6.91(dd,J=1.51 Hz, 8.3 Hz, 1 H), 6.82–6.78 (m, 2 H), 6.54 (s, 2 H), 6.48 (d, J =2.26 Hz, 2 H), 5.91 (d, J = 7.55 Hz, 1 H), 3.94 (s, 3 H), 3.90 (s, 3 H),3.85(s, 3 H),3.70 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3): d= 190.2,152.9, 150.1, 147.4, 142.1, 137.08, 135.9, 132.7, 132.3, 129.8, 129.5,129.3, 129.1, 124.6, 118.8, 113.6, 113.1, 110.5, 109.3, 105.7, 94.2,60.9, 55.9, 55.5 ppm; IR (KBr): nmax = 3444, 3341, 2934, 2836, 1629,1587, 1551, 1505, 1474, 1363, 1325, 1275, 1204, 1177, 1127, 1022,

878, 854, 776, 657, 591 cm�1; MS (ESI): m/z : 491 [M+H]+ ; HRMS(ESI): m/z : calcd for C28H31O6N2 : 491.21766 [M+H]+ ; found:491.21770.

(Z)-3-({2-Methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-1-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one (3 f): Compound 3 fwas prepared according to the method described for compound3 a by employing 1-[4-(trifluoromethoxy)phenyl]prop-2-yn-1-one(13 e, 67.88 mg, 0.317 mmol) to obtain pure 3 f as a yellow solid.Yield: 120 mg, 72 %; m.p. 103–105 8C; 1H NMR (300 MHz, CDCl3):d= 12.15 (d, J = 13.4 Hz, 1 H), 7.99 (d, J = 7.17 Hz, 2 H), 7.26 (s, 3 H),7.14 (s, 1 H), 6.97 (d, J = 8.49 Hz, 1 H), 6.82 (d, J = 8.49 Hz, 1 H), 6.50(d, J = 10.09 Hz, 4 H), 5.95 (d, J = 7.55 Hz, 1 H), 3.96 (s, 3 H), 3.85 (s,3 H), 3.71 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3): d= 189.03, 153.0,147.6, 143.6, 137.6, 132.7, 130.0, 129.9, 129.4, 129.1, 129.0, 124.8,120.3, 113.6, 110.7, 105.8, 93.9, 60.9, 55.9, 56.0 ppm; IR (KBr): nmax =3432, 2935, 2833, 1628, 1592, 1558, 1508, 1472, 1454, 1434, 1416,1363, 1325, 1298, 1279, 1257, 1180, 1129, 1037, 1015, 1006, 963,866, 844, 795, 779, 723 cm�1; MS (ESI): m/z : 530 [M+H]+ ; HRMS(ESI): m/z : calcd for C28H27O6NF3 : 530.17850 [M+H]+ ; found:530.17740.

tert-Butyl-3-[(Z)-3-({2-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phe-nyl}amino)acryloyl]-1H-indole-1-carboxylate (15 a): Compound15 a was prepared according to the method described for com-pound 3 a by employing tert-butyl 3-propioloyl-1H-indole-1-carbox-ylate (13 f ; 85.4 mg, 0.317 mmol) to obtain pure 15 a as a yellowsolid.

(Z)-1-(1H-indol-3-yl)-3-({2-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]-phenyl}amino)prop-2-en-1-one (3 g): TFA (0.01 mL, 0.188 mmol)was added to a stirred solution of 15 a (100 mg, 0.171 mmol) inCH2Cl2 at 0 8C. Then, the temperature of the mixture was slowly in-creased to RT, and the mixture was stirred for 2 h. The progress ofthe reaction was monitored by TLC (EtOAc/hexane = 7:3). The mix-ture was neutralized by adding a saturated solution of NaHCO3.The mixture was filtered through Celite, and the aqueous layer wasextracted with CHCl3. The organic layer was washed with brine,dried with anhydrous Na2SO4, and concentrated under reducedpressure to afford the crude compound. Yield: 60 mg, 72 %; m.p.95–97 8C; 1H NMR (300 MHz, CDCl3): d= 11.87 (d, J = 12.3 Hz, 1 H),8.62 (s, 1 H), 8.45–8.42 (m, 1 H), 7.80 (d, J = 2.8 Hz, 1 H), 7.42–7.39(m, 1 H), 7.30–7.27 (m, 1 H), 7.13–7.05 (m, 2 H), 6.9 (dd, J = 8.3 Hz,1.7 Hz, 1 H), 6.82 (d, J = 8.3 Hz, 1 H), 6.56 (s, 2 H), 6.50 (d, J = 4.7 Hz,2 H), 5.85 (d, J = 7.9 Hz, 1 H), 3.95(s, 3 H), 3.86 (s, 3 H), 3.71 ppm (s,6 H); 13C NMR (75 MHz, CDCl3): d= 188.1, 152.9, 147.3, 140.6, 136.5,132.8, 129.86, 129.83, 129.5, 128.9, 125.5, 123.6, 122.9, 122.0, 121.7,118.7, 112.8, 111.5, 110.5, 105.8, 96.4, 60.9, 55.9, 55.8 ppm; IR (KBr):nmax = 3388, 2953, 2859, 1628, 1580, 1513, 1453, 1428, 1366, 1324,1276, 1241, 1157, 1126, 1025, 1000, 966, 864, 790, 769, 758, 656,579 cm�1; MS (ESI): m/z : 485 [M+H]+ ; HRMS (ESI): m/z : calcd forC29H29O5N2 : 485.20711 [M+H]+ ; found: 485.20611.

(Z)-3-({2-Methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-1-(1-methyl-1H-indol-3-yl)prop-2-en-1-one (3 h): Compound 3 h wasprepared according to the method described for compound 3 a byemploying 1-(1-methyl-1H-indol-3-yl)prop-2-yn-1-one (13 g ;58.1 mg, 0.317 mmol) to obtain pure 3 h as a yellow solid. Yield:115 mg, 73 %; m.p. 122–124 8C; 1H NMR (300 MHz, CDCl3): d=11.87(d, J=12.1 Hz, 1 H), 8.45–8.41 (m, 1 H), 7.67(s, 1 H), 7.35–7.23(m, 3 H), 7.11–7.03 (s, 2 H), 6.92 (d, J = 8.3 Hz, 1 H), 6.78 (d, J =8.3 Hz, 1 H), 6.58 (s, 2 H), 6.49 (qt, J = 12.1 Hz, 5.3 Hz, 2 H), 5.79 (d,J = 7.55 Hz, 1 H), 3.96 (s, 3 H), 3.83 (s, 6 H), 3.70 ppm (s, 6 H);13C NMR (75 MHz, CDCl3): d= 187.5, 152.9, 147.3, 140.3, 137.4,133.2, 132.8, 130.0, 129.8, 129.5, 128.9, 126.3, 123.4, 122.7, 122.4,121.7, 112.7, 110.4, 109.4, 105.7, 96.4, 61.0, 60.9, 55.9 ppm; IR (KBr):




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nmax = 3434, 2929, 2835, 1631, 1604, 1578, 1524, 1488, 1469, 1433,1415, 1368, 1327, 1277, 1217, 1187, 1146, 1126, 1087, 1026, 1005,947, 877, 857, 783, 752 cm�1; MS (ESI): m/z : 499 [M+H]+ ; HRMS(ESI): m/z : calcd for C30H31O5N2 : 499.22275 [M+H]+ ; found:499.22339.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-no)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4 a): Compound4 a was prepared according to the method described for com-pound 3 a by employing 12 b (100 mg, 0.289 mmol) and 13 a(63.8 mg, 0.289 mmol) to obtain pure 4 a as a yellow solid. Yield:120 mg, 73 %; m.p. 61–63 8C; 1H NMR (300 MHz, CDCl3): d= 12.12(d, J = 12.1 Hz, 1 H), 7.30–7.23 (m, 1 H), 7.19 (s, 2 H), 6.79 (s, 1 H),6.58–6.49 (m, 5 H), 5.93 (d, J = 8.3 Hz, 1 H), 3.96 (s, 3 H), 3.94 (s, 6 H),3.90 (s, 3 H), 3.84 (s, 3 H), 3.82 (s, 3 H), 3.72 ppm (s, 6 H); 13C NMR(75 MHz, CDCl3): d= 189.7, 152.7, 152.5, 142.9, 140.8, 137.05, 136.6,134.64, 134.6, 133.6, 133.2, 130.1, 129.2, 128.4, 127.4, 107.6, 106.2,105.7, 104.4, 103.3, 94.06, 60.7, 60.6, 56.9, 55.75, 55.58 ppm; IR(KBr): nmax = 3424, 2935, 2832, 1629, 1585, 1554, 1503, 1470, 1425,1367, 1327, 1277, 1235, 1200, 1165, 1125, 1075, 1000, 913, 862,838, 772, 734, 659, 585 cm�1; MS (ESI): m/z : 566 [M+H]+ ; HRMS(ESI): m/z : calcd for C31H36O9N: 566.23846 [M+H]+ ; found:566.23749.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-({2,3-dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (4 b): Com-pound 4 b was prepared according to the method described forcompound 3 a by employing 12 b (100 mg, 0.289 mmol) and 13 b(86.5 mg, 0.289 mmol) to obtain pure 4 b as a yellow solid. Yield:130 mg, 69.7 %; m.p. 55–57 8C; 1H NMR (300 MHz, CDCl3): d= 12.04(d, J = 12.8 Hz, 1 H), 7.31–7.24 (m, 1 H), 6.87 (s, 1 H), 6.76 (d, J =6.7 Hz, 1 H), 6.57 (s, 1 H), 6.52–6.47 (m, 4 H), 5.67 (d, J = 7.55 Hz, 1 H),3.96 (s, 3 H), 3.90 (d, J = 3.02 Hz, 9 H), 3.87 (s, 3 H), 3.71 ppm (s, 9 H);13C NMR (75 MHz, CDCl3): d= 192.4, 152.9, 152.7, 150.8, 144.1,142.9, 138.3, 136.8, 133.6, 133.4, 132.3, 130.4, 129.3, 108.0, 107.9,106.5, 106.1, 105.9, 98.6, 61.12, 60.8, 60.9, 55.7, 55.9, 56.1 ppm; IR(KBr): nmax = 3434, 2935, 2833, 1630, 1582, 1505, 1466, 1423, 1381,1329, 1276, 1239,1203, 1166, 1126, 1104, 1075, 1005, 932, 864, 850,794, 657 cm�1; MS (ESI): m/z : 644 [M+H]+ ; HRMS (ESI): m/z : calcdfor C31H35O9NBr: 644.14897 [M+H]+ ; found: 644.14957.

(Z)-1-[3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl]-3-({2,3-di-methoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (14 b): Compound 14 b was prepared according to themethod described for compound 3 a by employing 12 b (100 mg,0.289 mmol) and 13 c (83.9 mg, 0.289 mmol) to obtain pure 14 b asa yellow solid. Yield: 150 mg, 81 %.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-no)-1-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (4 c): Com-pound 4 c was prepared according to the method described forcompound 3 c by employing 14 b (150 mg, 0.236 mmol) and TBAF(1 n in THF) (0.3 mL, 0.26 mmol) to obtain pure 4 c as a yellowsolid. Yield: 99 mg, 80 %; m.p. 64–66 8C; 1H NMR (300 MHz, CDCl3):d= 12.09 (d, J = 12.8 Hz, 1 H), 7.53 (d, J = 6.7 Hz, 2 H), 7.29–7.22 (m,1 H), 6.89 (d, J = 9.06 Hz, 1 H), 6.75 (s, 1 H), 6.53–6.51 (m, 5 H), 5.94(d, J = 8.3 Hz, 1 H), 5.78 (s, 1 H), 3.95 (s, 3 H), 3.94 (s, 3 H), 3.83 (s,3 H), 3.71 ppm (s, 9 H), 13C NMR (75 MHz, CDCl3): d= 189.9, 152.9,152.7, 149.4, 145.2, 142.6, 137.2, 136.7, 134.1, 133.3, 132.9, 132.4,130.2, 129.4, 120.4, 113.6, 109.8, 107.4, 106.4, 105.9, 94.2, 60.8, 60.7,55.9, 55.7 ppm; IR (KBr): nmax = 3331, 2990, 2965, 2935, 2837, 1632,1580, 1546, 1504, 1464, 1435, 1370, 1328, 1282, 1203, 1178, 1162,1127, 1074, 1054, 1026, 999, 974,945, 853, 775, 691 cm�1; MS (ESI):m/z : 522 [M+H]+ ; HRMS (ESI): m/z : calcd for C29H32O8N: 522.21224[M+H]+ ; found: 522.21105.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-no)-1-(4-methoxy-3-nitro phenyl)prop-2-en-1-one (4 d): Com-pound 4 d was prepared according to the method described forcompound 3 a by employing 12 b (100 mg, 0.289 mmol) and 13 d(59.3 mg, 0.289 mmol) to obtain pure 4 d as a yellow solid. Yield:120 mg, 75 %; m.p. 144–147 8C; 1H NMR (300 MHz, CDCl3): d= 12.21(d, J = 12.8 Hz, 1 H), 8.43 (s, 1 H), 8.14 (d, J = 9.06 Hz, 1 H), 7.37–7.30(m, 1 H), 7.12 (d, J = 9.06 Hz, 1 H), 6.77 (s, 1 H), 6.57–6.47 (m, 5 H),5.92 (d, J = 7.5 Hz, 1 H), 4.03 (s, 3 H), 3.97 (s, 3 H), 3.84 (s, 3 H),3.74 ppm (s, 9 H); 13C NMR (75 MHz, CDCl3): d= 187.1, 155.0, 152.9,143.9, 139.1, 137.2, 136.9, 133.5, 133.4, 133.1, 131.6, 130.4, 129.2,125.06, 113.06, 108.0, 106.5, 105.8, 93.3, 60.9, 60.8, 56.7, 55.9,55.7 ppm; IR (KBr): nmax = 3433, 2963, 2937, 2838, 1636, 1592, 1556,1503, 1464, 1429, 1420, 1324, 1295, 1282, 1235, 1128, 1075, 1013,999, 926 cm�1; MS (ESI): m/z : 551 [M+H]+ ; HRMS (ESI): m/z : calcdfor C30H31O9N2 : 551.2029 [M+H]+ ; found: 551.2031.

(Z)-1-(3-Amino-4-methoxyphenyl)-3-({2,3-dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (4 e): Compound4 e was prepared according to the method described for com-pound 3 e by employing 4 d (150 mg, 0.288 mmol), zinc (72.71 mg,1.15 mmol), and ammonium formate (73.67 mg, 1.15 mmol) toobtain pure 4 e as a yellow solid. Yield: 111 mg, 78 %; m.p. 68–71 8C; 1H NMR (300 MHz, CDCl3): d= 12.1 (d, J = 12.1 Hz, 1 H), 7.34(d, J = 7.4 Hz, 2 H), 7.23–7.19 (m, 1 H), 6.81 (d, J = 8.49 Hz, 1 H), 6.75(s, 1 H), 6.53–6.51 (m, 5 H), 5.96 (d, J = 7.9 Hz, 1 H), 3.95 (s, 3 H), 3.90(s, 3 H), 3.83 (s, 3 H), 3.72 ppm (s, 9 H); 13C NMR (75 MHz, CDCl3): d=190.4, 152.9, 152.7, 150.1, 142.2, 137.1, 136.6, 135.9, 134.1, 133.3,132.4, 132.3, 130.1, 129.4, 118.8, 113.6, 109.2, 107.2, 106.3, 105.8,94.4, 60.8, 60.7, 55.9, 55.7, 55.5 ppm; IR (KBr): nmax = 3444, 3371,2933, 2834, 1630, 1584, 1552, 1504, 1469, 1428, 1367, 1326, 1276,1204, 1176, 1146, 1126, 1076, 999, 919, 854, 774 cm�1; MS (ESI): m/z : 521 [M+H]+ ; HRMS (ESI): m/z : calcd for C29H33O7N2 : 521.22823[M+H]+ ; found: 521.22722.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-no)-1-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one (4 f): Com-pound 4 f was prepared according to the method described forcompound 3 a by employing 12 b (100 mg, 0.289 mmol) and 13 e(61.9 mg, 0.289 mmol) to obtain pure 4 f as a yellow solid. Yield:120 mg, 74 %; m.p. 107–110 8C; 1H NMR (300 MHz, CDCl3): d= 12.21(d, J = 12.8 Hz, 1 H), 7.98 (d, J = 8.3 Hz, 2 H), 7.35–7.31 (m, 1 H), 7.27–7.25 (m, 3 H), 6.77 (s, 1 H), 6.57–6.46 (m, 4 H), 5.93 (d, J = 8.3 Hz,1 H), 3.96 (s, 3 H), 3.83 (s, 3 H), 3.71 ppm (s, 9 H); 13C NMR (75 MHz,CDCl3): d= 182.9, 152.9, 152.7, 151.4, 143.7, 137.6, 137.2, 136.9,133.6, 133.4, 132.3, 132.0, 130.3, 129.3, 129.1, 128.0, 120.9, 107.9,106.5, 105.9, 94.03, 60.89, 60.8, 55.9, 55.7 ppm; IR (KBr): nmax = 3432,2947, 2842, 1621, 1534, 1514, 1501, 1450, 1411, 1321, 1315, 1292,1255, 1189, 1165, 1117, 1052, 1001, 925, 845, 818, 789 cm�1; MS(ESI): m/z : 560 [M+H]+ ; HRMS (ESI): m/z : calcd for C29H29O7NF3:560.18906 [M+H]+ ; found: 560.18821.

tert-Butyl 3-[(Z)-3-({2,3-dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]-phenyl}amino)acryloyl]-1H-indole-1-carboxylate (15 b): Com-pound 15 b was prepared according to the method described forcompound 3 a by employing 13 f (77.8 mg, 0.289 mmol) to obtainpure 15 b as a yellow solid.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-no)-1-(1H-indol-3-yl)prop-2-en-1-one (4 g): Compound 4 g wasprepared according to the method described for compound 3 g byemploying 15 b (100 mg, 0.163 mmol) and TFA (0.01 mL,0.178 mmol) to obtain pure 4 g as a yellow solid. Yield: 62 mg,74 %; m.p. 88–90 8C; 1H NMR (300 MHz, CDCl3): d= 11.96 (d, J =11.9 Hz, 1 H), 8.47 (s, 1 H), 8.37–8.36 (m, 1 H), 7.82 (d, J = 2.89 Hz,




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1 H), 7.44–7.39 (m, 2 H), 7.32–729 (m, 1 H), 7.15 (dd, J = 4.1 Hz,8.1 Hz, 1 H), 6.75 (d, J = 1.2 Hz, 1 H), 6.53 (d, J = 12.1 Hz, 4 H), 5.86(d, J = 8.1 Hz, 1 H), 3.96 (s, 3 H), 3.84 (s, 3 H), 3.72 (s, 3 H), 3.71 ppm(s, 6 H); IR (KBr): nmax = 3415, 2931, 2839, 1627, 1580, 1516, 1427,1372, 1310, 1275, 1239, 1158, 1125, 1078, 1036, 1007, 997, 889,771 cm�1; MS (ESI): m/z : 515 [M+H]+ ; HRMS (ESI): m/z : calcd forC30H31O6N2 : 515.21766 [M+H]+ ; found: 515.21847.

(Z)-3-({2,3-Dimethoxy-5-[(Z)-3,4,5-trimethoxystyryl]phenyl}ami-no)-1-(1-methyl-1H-indol-3-yl)prop-2-en-1-one (4 h): Compound4 h was prepared according to the method described for com-pound 3 a by employing 13 g (52.9 mg, 0.289 mmol) to obtainpure 4 h as a yellow solid. Yield: 112 mg, 73 %; m.p. 112–115 8C;1H NMR (300 MHz, CDCl3): d= 12.01 (d, J = 12.1 Hz, 1 H), 8.50 (s,1 H), 7.73 (s, 1 H), 7.37 (s, 3 H), 7.24–7.17 (m, 1 H), 6.8 (s, 1 H), 6.59 (d,J = 12.1 Hz, 5 H), 5.90 (d, J = 7.55 Hz, 1 H), 4.05 (s, 3 H), 3.92 (s, 3 H),3.87 (s, 3 H), 3.72 ppm (s, 9 H); 13C NMR (75 MHz, CDCl3): d= 187.5,153.02, 140.5, 137.5, 134.8, 133.3, 133.1, 132.5, 132.0, 129.6, 126.4,122.7, 122.4, 121.8, 117.8, 109.4, 107.0, 106.3, 106.1, 96.7, 61.03,60.9, 60.7, 56.02, 55.8 ppm; IR (KBr): nmax = 3432, 2931, 2832, 1628,1528, 1522, 1505, 1487, 1469, 1425, 1366, 1326, 1276, 1216, 1147,1125, 1087, 999, 947, 859, 791, 770 cm�1; MS (ESI): m/z : 529[M+H]+ ; HRMS (ESI): m/z : calcd for C31H33O6N2 : 529.23331 [M+H]+ ;found: 529.23274.

3-Hydroxy-4-methoxy-2-nitrobenzaldehyde (17): Nitromethane(20 mL) and nitronium tetrafluoroborate (9.2 g, 0.069 mol) wereadded to a stirred solution of isovaniline (16 ; 10 g, 0.066 mol) inCH2Cl2 (30 mL) at �40 8C. Then, the mixture was stirred for 6 h. Theprogress of the reaction was monitored by TLC (EtOAc/hexane =2:8). Water was added to the mixture at RT. The mixture was thenconcentrated, and the aqueous layer was extracted with diethylether (3 �). The organic layer was washed with water and brine,dried with anhydrous Na2SO4, and concentrated under reducedpressure to afford the crude compound, which was purified bycolumn chromatography (EtOAc/hexane = 3:7). Yield: 6.0 g, 46 %;1H NMR (300 MHz, CDCl3): d= 9.79 (s, 1 H), 7.39 (d, J = 8.3 Hz, 1 H),7.08 (d, J = 8.4 Hz, 1 H), 4.02 ppm (s, 3 H); MS (ESI): m/z : 220[M+Na]+ .

3-(tert-Butyldimethylsilyloxy)-4-methoxy-2-nitrobenzaldehyde(18): Imidazole (0.789 g, 0.0116 mol) and tert-butylchlorodimethylsi-lane (1.68 g, 0.0116 mol) were added to a stirred solution of 17(2.0 g, 0.0101 mol) in CH2Cl2 (30 mL) at 0 8C. The temperature wasslowly increased to RT, and the mixture was stirred for 3 h. Theprogress of the reaction was monitored by TLC (EtOAc/hexane =2:8). Upon completion of the reaction, water was added to mix-ture, which was extracted with CH2Cl2. The organic layer waswashed with water and brine and dried with anhydrous Na2SO4. Fi-nally, the solvent was evaporated under reduced pressure to affordthe pure compound. Yield: 2.9 g, 91 %; 1H NMR (300 MHz, CDCl3):d= 9.56 (s, 1 H), 7.26 (d, J = 8.31 Hz, 1 H), 6.82 (d, J = 8.31 Hz, 1 H),3.74 (s, 3 H), 0.93 (s, 9 H), 0.09 ppm (s, 6 H); MS (ESI): m/z : 312[M+H]+ .

(Z)-tert-Butyl[6-methoxy-2-nitro-3-(3,4,5-trimethoxystyryl)phe-noxy]dimethylsilane (19): Compound 19 was prepared accordingto the method described for compound 11 a by employing 18(3.69 g, 7.06 mmol) to obtain pure 19 as a yellow solid. Yield: 2.7 g,48 %; 1H NMR (300 MHz, CDCl3): d= 6.77 (s, 2 H), 6.61 (d, J = 12.0 Hz,1 H), 6.41 (s, 2 H), 6.38 (d, J = 12.0 Hz, 1 H), 3.82 (s, 3 H), 3.81 (s, 3 H),3.67 (s, 6 H), 0.94 (s, 9 H), 0.21 ppm (s, 6 H). MS (ESI, m/z): 476[M+1]+ .

(Z)-2-(tert-Butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethox-ystyryl)aniline (20): Compound 20 was prepared according to the

method described for compound 12 a by employing 19 (900 mg,1.89 mmol) and zinc (484.9 mg, 7.57 mmol) to obtain pure 20 asa light brown solid. Yield: 700 mg, 83 %; 1H NMR (300 MHz, CDCl3):d= 6.70 (d, J = 8.3 Hz, 1 H), 6.51 (s, 2 H), 6.47 (d, J = 4.5 Hz, 2 H), 6.27(d, J = 8.3 Hz, 1 H), 3.81 (s, 3 H), 3.76 (s, 3 H), 3.63 (s, 6 H), 1.02 (s,9 H), 0.17 ppm (s, 6 H); MS (ESI): m/z : 446 [M+H]+ .

(Z)-2-Amino-6-methoxy-3-(3,4,5-trimethoxystyryl)phenol (21):Compound 21 was prepared according to the method describedfor compound 3 c by employing 20 (100 mg, 0.224 mmol) toobtain pure 21 as a yellow liquid. 1H NMR (300 MHz, CDCl3): d=6.67 (d, J = 8.3 Hz, 1 H), 6.53–6.43 (m, 4 H), 6.32 (d, J = 8.3 Hz, 1 H),3.85 (s, 3 H), 3.81 (s, 3 H), 3.63 ppm (s, 6 H); MS (ESI): m/z : 332[M+H]+ .

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-l}amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5 a): Com-pound 5 a was prepared according to the method described forcompound 3 a by employing 21 (100 mg, 0.301 mmol) and 13 a(66.2 mg, 0.301 mmol) to obtain pure 5 a as a yellow solid. Yield:123 mg, 73 %; m.p. 61–63 8C; 1H NMR (300 MHz, CDCl3): d= 11.93(d, J = 12.2 Hz, 1 H), 7.84–7.75 (m, 1 H), 7.18 (s, 2 H), 6.80 (d, J =8.12 Hz, 1 H), 6.67 (d, J = 11.8 Hz, 1 H), 6.61–6.55 (m, 2 H), 6.44 (s,2 H), 5.85–5.81 (m, 2 H), 3.92 (s, 6 H), 3.91 (s, 3 H), 3.90 (s, 3 H), 3.81(s, 3 H), 3.58 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3): d= 189.6, 152.8,152.6, 149.8, 146.3, 137.2, 137.0, 135.0, 132.3, 132.0, 125.6, 125.2,122.3, 121.1, 106.1, 105.9, 104.5, 92.7, 60.89, 60.8, 56.2, 56.1,55.6 ppm; IR (KBr): nmax = 3411, 2953, 2925, 2852, 1625, 1583, 1550,1496, 1463, 1410, 1327, 1286, 1201, 1166, 1125, 1088, 1002, 919,855, 833, 773 cm�1; MS (ESI): m/z : 552 [M+H]+ ; HRMS (ESI): m/z :calcd for C30H34O9N: 552.22281 [M+H]+ ; found: 552.22399.

(Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-({2-hydroxy-3-me-thoxy-6-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (5 b): Compound 5 b was prepared according to the methoddescribed for compound 3 a by employing 13 b (89.6 mg,0.301 mmol) to obtain pure 5 b as a yellow solid. Yield: 135 mg,74 %; m.p. 54–56 8C; 1H NMR (300 MHz, CDCl3): d= 11.86 (d, J =12.6 Hz, 1 H), 7.93–7.84 (m, 1 H), 6.85–6.80 (m, 2 H), 6.68 (d, J =11.89 Hz, 1 H), 6.61–6.55 (m, 2 H), 6.46 (s, 2 H), 5.89 (s, 1 H), 5.54 (d,J = 7.55 Hz, 1 H), 3.90 (s, 6 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.81 (s, 3 H),3.63 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3): d= 199.1, 152.5, 150.8,149.6, 146.3, 143.9, 138.4, 136.9, 132.6, 131.9, 125.3, 124.9, 122.2,121.1, 108.0, 106.1, 106.0, 97.04, 61.1, 60.9, 60.8, 56.3, 56.1,55.7 ppm; IR (KBr): nmax = 3425, 2957, 2935, 2836, 1625, 1580, 1504,1461, 1423, 1381, 1330, 1285, 1204, 1164, 1126, 1104, 1006, 850,790 cm�1; MS (ESI): m/z : 630 [M+H]+ ; HRMS (ESI): m/z : calcd forC30H33O9NBr: 630.13332 [M+H]+ ; found: 630.13385.

(Z)-1-[3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl]-3-({2-hy-droxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)-prop-2-en-1-one (14 c): Compound 14 c was prepared accordingto the method described for compound 3 a by employing 13 c(82.4 mg, 0.301 mmol) to obtain pure 14 c as a yellow solid. Yield:138 mg, 73 %.

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-l}amino)-1-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (5 c):Compound 5 c was prepared according to the method describedfor compound 3 c by employing 14 c (100 mg, 0.161 mmol) toobtain pure 5 c as a yellow solid. Yield: 63 mg, 76 %; m.p. 65–67 8C;1H NMR (300 MHz, CDCl3): d= 11.95 (d, J = 12.08 Hz, 1 H), 7.82–7.73(m, 1 H), 7.52–7.48 (m, 2 H), 6.86 (d, J = 8.3 Hz, 1 H), 6.79 (d, J =8.3 Hz, 1 H), 6.67 (d, J = 12.08 Hz, 1 H), 6.58–6.54 (m, 2 H), 6.44 (s,2 H), 5.84–5.81 (m, 2 H), 5.64 (s, 1 H), 3.95 (s, 3 H), 3.94 (s, 3 H), 3.90(s, 3 H), 3.81 (s, 3 H), 3.59 ppm (s, 6 H), 13C NMR (75 MHz, CDCl3): d=




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189.6, 152.5, 149.4, 146.3, 145.2, 137.05, 136.9, 132.9, 132.1, 132.07,125.8, 125.2, 122.1, 120.9, 120.2, 113.6, 109.8, 105.9, 105.8, 92.660.7, 56.2, 55.8, 55.6 ppm; IR (KBr): nmax = 3417, 2959, 2935, 2837,1626, 1608, 1587, 1548, 1496, 1462, 1421, 1327, 1265, 1125, 1008,1020, 849, 775 cm�1; MS (ESI): m/z : 508 [M+H]+ ; HRMS (ESI): m/z :calcd for C28H30O8N: 508.19659 [M+H]+ ; found: 508.19559.

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-l}amino)-1-(4-methoxy-3-nitrophenyl)prop-2-en-1-one (5 d): Com-pound 5 d was prepared according to the method described forcompound 3 a by employing 13 d (61.7 mg, 0.301 mmol) to obtainpure 5 d as a yellow solid. Yield: 124 mg, 76 %; m.p. 120–124 8C;1H NMR (500 MHz, CDCl3): d= 11.98 (d, J = 12.5 Hz, 1 H), 8.42 (d, J =2.1 Hz, 1 H), 8.14–8.12 (m, 1 H), 7.88–7.84 (m, 1 H), 7.11 (d, J =8.85 Hz, 1 H), 6.81 (d, J = 8.85 Hz, 1 H), 6.69 (d, J = 11.9 Hz, 1 H), 6.61(d, J = 8.3 Hz, 1 H), 6.56 (d, J = 11.7 Hz, 1 H), 6.43 (s, 2 H), 5.88 (s, 1 H),5.80 (d, J = 7.7 Hz, 1 H), 4.01 (s, 3 H), 3.91 (s, 3 H), 3.80 (s, 3 H),3.59 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3): d= 186.6, 154.8, 152.6,150.6, 146.3, 139.1, 137.2, 137.0, 133.0,132.6, 131.9, 125.3, 124.9,122.4, 121.12, 112.9, 106.4, 105.9, 91.9, 60.8, 56.6, 56.3, 55.6 ppm; IR(KBr): nmax = 3400, 2955, 2937, 2863, 1627, 1592, 1556, 1496, 1463,1421, 1328, 1258, 1163, 1126, 1087, 1041, 1013, 963, 922, 855, 776,736 cm�1; MS (ESI): m/z : 537 [M+H]+ ; HRMS (ESI): m/z : calcd forC28H29O9N2 : 537.18676 [M+H]+ ; found: 537.18637.

(Z)-1-(3-Amino-4-methoxyphenyl)-3-({2-hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]phenyl}amino)prop-2-en-1-one (5 e):Compound 5 e was prepared according to the method describedfor compound 3 e by employing 5 d (100 mg, 0.186 mmol) toobtain pure 5 e as a yellow solid. Yield: 70 mg, 74 %; m.p. 75–78 8C;1H NMR (300 MHz, CDCl3): d= 12.08 (d, J = 12.08 Hz, 1 H), 7.36–7.33(m, 2 H), 7.23–7.19 (m, 1 H), 6.82–6.75 (m, 2 H), 6.51 (d, J = 5.28 Hz,6 H), 5.95 (d, J = 7.93 Hz, 1 H), 3.95 (s, 3 H), 3.90 (s, 3 H), 3.83 (s, 3 H),3.71 ppm (s, 6 H); IR (KBr): nmax = 3426, 2995, 2938, 2837, 1610,1579, 1532, 1505, 1462, 1421, 1353, 1327, 1283, 1237, 1185, 1126,1089, 1009, 855, 827, 774 cm�1; MS (ESI): m/z : 507 [M+H]+ ; HRMS(ESI): m/z : calcd for C28 H31O7N2 : 507.21258 [M+H]+ ; found:507.21359.

(Z)-3-({2-Hydroxy-3-methoxy-6-[(Z)-3,4,5-trimethoxystyryl]pheny-l}amino)-1-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one (5 f):Compound 5 f was prepared according to the method describedfor compound 3 a by employing 13 e (64.4 mg, 0.301 mmol) toobtain pure 5 f as a yellow solid. Yield: 130 mg, 78 %; m.p. 97–99 8C; 1H NMR (300 MHz, CDCl3): d= 12.01 (d, J = 12.8 Hz, 1 H), 7.96(d, J = 8.3 Hz, 2 H), 7.87–7.80 (m, 1 H), 7.23 (d, J = 8.3 Hz, 3 H), 6.79(d, J = 8.3 Hz, 1 H), 6.65 (d, J = 11.3 Hz, 1 H), 6.60–6.51 (m, 2 H), 6.43(s, 2 H), 5.79 (d, J = 7.5 Hz, 1 H), 3.85 (s, 3 H), 3.80 (s, 3 H), 3.56 ppm(s, 6 H); 13C NMR (75 MHz, CDCl3): d= 188.7, 152.5, 151.2, 150.5,146.3, 137.8, 137.2, 137.1, 132.5, 132.01, 129.02, 125.4, 125.05,122.4, 122.01, 121.07, 120.23, 106.3, 105.9, 105.7, 92.5, 60.7, 56.2,55.6 ppm; IR (KBr): nmax = 3429, 3311, 2935, 2898, 2837, 1625, 1606,1550, 1503, 1462, 1421, 1327, 1284, 1257, 1176, 1126, 1088, 1007,855, 775 cm�1; MS (ESI): m/z : 546 [M+H]+ ; HRMS (ESI): m/z : calcdfor C28H27O7NF3 : 546.1739 [M+H]+ ; found: 546.1766.

General procedure for the preparation of 1-arylprop-2-yn-1-one(13): 0.5 n Ethynylmagnesium bromide in THF (1.5 mmol) wasadded to a stirred solution of substituted benzaldehyde 22 a–g(1 mmol) in THF at 0 8C. Then, the temperature of the mixture wasslowly increased to RT, and the mixture was stirred for 6–8 h. Theprogress of the reaction was monitored by TLC (EtOAc/hexane =1:1). A solution of saturated ammonium chloride was added, andthe mixture was concentrated and extracted with EtOAc. The or-ganic layer was washed with brine, dried with anhydrous Na2SO4,

and concentrated under reduced pressure to afford pure 23 a–g.Compound 23 a–g (1 mmol) was dissolved in DMSO, and a solutionof IBX (1.1 mmol) in DMSO (10 mL) was added at 10–15 8C. Then,the temperature of the mixture was slowly increased to RT, andthe mixture was stirred for 4–6 h. The progress of the reaction wasmonitored by TLC (EtOAc/hexane = 3:7). Water was added, and themixture was filtered through Celite. The aqueous layer was extract-ed with EtOAc. The organic layer was washed with water andbrine, dried with anhydrous Na2SO4, and concentrated under re-duced pressure to afford the crude product, which was purified bycolumn chromatography (EtOAc/hexane = 3:7).

1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one (13 a): Compound13 a was prepared according to the method described for com-pound 13 by employing 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-ol(23 a ; 750 mg, 3.38 mmol) and IBX (1.04 g, 3.7 mmol) to obtainpure 13 a as a light brown solid. Yield: 650 mg, 88 %; m.p. 123–125 8C; 1H NMR (300 MHz, CDCl3): d= 7.43 (s, 2 H), 3.95 (s, 3 H), 3.93(s, 6 H), 3.43 ppm (s, 1 H); MS (ESI): m/z : 221 [M+H]+ .

1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (13 b): Com-pound 13 b was prepared according to the method described forcompound 13 by employing 1-(2-bromo-3,4,5-trimethoxyphenyl)-prop-2-yn-1-ol (23 b ; 750 mg, 2.49 mmol) and IBX (767 mg,2.74 mmol) to obtain pure 13 b as a brown solid. Yield: 640 mg,86 %; m.p. 79–80 8C; 1H NMR (300 MHz, CDCl3): d= 7.46 (s, 1 H), 3.98(s, 3 H), 3.93 (s, 3 H), 3.89 (s, 3 H), 3.50 ppm (s, 1 H); MS (ESI): m/z :298 [M+H]+ .

1-[4-(tert-Butyldimethylsilyloxy)-3-methoxyphenyl]prop-2-yn-1-one (13 c): Compound 13 c was prepared according to the methoddescribed for compound 13 by employing 1-[3-(tert-butyldimethyl-silyloxy)-4-methoxyphenyl]prop-2-yn-1-ol (23 c ; 750 mg, 2.57 mmol)and IBX (790 mg, 2.82 mmol) to obtain pure 13 c as a brown solid.Yield: 620 mg, 83 %; m.p. 80–82 8C; 1H NMR (300 MHz, CDCl3): d=7.67 (dd, J = 6.41 Hz, 0.92 Hz, 1 H), 7.43 (dd, J = 0.92 Hz, 1.52 Hz,1 H), 6.73 (d, J = 8.54 Hz, 1 H), 3.71 (s, 3 H), 3.19 (s, 1 H), 0.83 (s, 9 H),0.12 ppm (s, 6 H); MS (EI): m/z : 291 [M+H]+ .

1-(3-Methoxy-4-nitrophenyl)prop-2-yn-1-one (13 d): Compound13 d was prepared according to the method described for com-pound 13 by employing 1-(4-methoxy-3-nitrophenyl)prop-2-yn-1-ol(23 d ; 750 mg, 3.62 mmol) and IBX (1.12 g, 3.98 mmol) to obtainpure 13 d as a brown solid. Yield: 500 mg, 74 %; m.p. 120–123 8C;1H NMR (500 MHz, CDCl3): d= 8.61 (d, J = 2.0 Hz, 1 H), 8.31 (dd, J =2.0 Hz, 8.85 Hz, 1 H), 7.19 (d, J = 8.85 Hz, 1 H), 4.06 (s, 3 H), 3.51 ppm(s, 1 H). MS (EI): m/z : 206 [M+H]+ .

1-[3-(Trifluoromethoxy)phenyl]prop-2-yn-1-one (13 e): Com-pound 13 e was prepared according to the method described forcompound 13 by employing 1-[4-(trifluoromethoxy)phenyl]prop-2-yn-1-ol (23 e ; 750 mg, 3.47 mmol) and IBX (1.07 g, 3.82 mmol) toobtain pure 13 e as a brown solid. Yield: 600 mg, 80 %; m.p. 97–99 8C; 1H NMR (500 MHz, CDCl3): d= 7.83 (d, J = 8.3 Hz, 2 H), 7.21 (d,J = 8.3 Hz, 2 H), 3.15 ppm (s, 1 H); MS (EI): m/z : 215 [M+H]+ .

tert-Butyl 3-propioloyl-1H-indole-1-carboxylate (13 f): Compound13 f was prepared according to the method described for com-pound 13 by employing tert-butyl 3-(1-hydroxyprop-2-yn-1-yl)-1H-indole-1-carboxylate (23 f ; 750 mg, 2.76 mmol) and IBX (851 mg,3.04 mmol) to obtain pure 13 f as a brown solid. Yield: 620 mg,83 %; m.p. 150–152 8C; 1H NMR (300 MHz, CDCl3): d= 8.43 (s, 1 H),8.32 (d, J = 8.1 Hz, 1 H), 8.12 (s, J = 8.1 Hz, 1 H), 7.41–7.35 (m, 2 H),3.27 (s, 1 H), 1.72 ppm (s, 9 H); MS (EI): m/z : 291 [M+H]+ .

1-(1-Methyl-1H-indol-3-yl)prop-2-yn-1-one (13 g): Compound 13 gwas prepared according to the method described for compound




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13 by employing 1-(1-methyl-1H-indol-3-yl)prop-2-yn-1-ol (23 g ;800 mg, 4.32 mmol) and IBX (1.33 g, 4.75 mmol) to obtain pure13 g as a brown solid. Yield: 800 mg, 73 %; m.p. 180–183 8C;1H NMR (300 MHz, CDCl3): d= 8.38–8.35 (m, 1 H), 7.95 (s, 1 H), 7.36(t, J = 2.83 Hz, 5.09 Hz, 2 H), 7.33–7.32 (m, 1 H), 3.88 (s, 3 H),3.16 ppm (s,1 H); MS (EI): m/z : 184 [M+H]+ .

Biology

Cell cultures, maintenance, and antiproliferative evaluation : All thecell lines used in this study were purchased from the AmericanType Culture Collection (ATCC, United States). A549, MCF-7, HeLa,and HCT116 were grown in Dulbecco’s modified Eagle’s medium(containing 10 % fetal bovine serum in a humidified atmosphere of5 % CO2 at 37 8C). Cells were trypsinized when subconfluent fromT25 flasks/60 mm dishes and seeded in 96-well plates. The amino-stilbene–arylpropenones were evaluated for their in vitro antiproli-ferative activity in four different human cancer cell lines. A protocolof 48 h continuous drug exposure was used, and a sulforhodami-ne B (SRB) cell proliferation assay was employed to estimate cell vi-ability or growth. The cell lines were grown in their respectivemedia containing 10 % fetal bovine serum and were seeded into96-well microtiter plates in 200 mL aliquots at plating densities de-pending on the doubling time of individual cell lines. The microtit-er plates were incubated at 37 8C, 5 % CO2, 95 % air, and 100 % rela-tive humidity for 24 h prior to the addition of the experimentaldrugs. Aliquots of 2 mL of the test compounds were added to thewells already containing 198 mL of cells, which resulted in the re-quired final drug concentrations. For each compound, four concen-trations (0.1, 1, 10, and 100 mm) were evaluated, and each wasdone in triplicate wells. Plates were incubated for another 48 h,and the assay was terminated by the addition of 100 mL of 10 % wt/vol cold trichloroacetic acid and incubated at 4 8C for 1 h. The su-pernatant was discarded. The plate was washed with tap water(4 �) and was allowed to air dry. The cells were then stained with0.057 % SRB dissolved in 1 % acetic acid for 30 min at RT. UnboundSRB was washed away with four washes of 1 % acetic acid. Theplate was again allowed to air dry, and the bound SRB stain, repre-senting surviving cells, was dissolved in Tris base (10 mm, 50 mL).The optical density was determined at l= 510 nm by using a micro-plate reader (Enspire, PerkinElmer, USA). Percent growth was calcu-lated on a plate-by-plate basis for test wells relative to controlwells. The above determinations were repeated thrice. The growthinhibitory effects of the compounds were analyzed by generatingdose response curves as a plot of the percentage surviving cellsversus compound concentration. The sensitivity of the cancer cellsto the test compound was expressed in terms of IC50, a value de-fined as the concentration of compound that produced 50 % re-duction relative to the control absorbance. IC50 values are indicatedas means� standard deviations of three independent experi-ments.[27]

Dot-blot assay : A549 cells were treated with 1 mm 3 a–h, 4 a–h, and5 a–f for 24 h. Subsequently, cells were harvested and proteinswere quantified by using Amido Black followed by densitometryanalysis. Equal amounts of protein were blotted on a nitrocellulosemembrane by using a Bio-Dot SF microfiltration apparatus (Bio-Rad). Briefly, the nitrocellulose membrane and three filters papers(Whatmann 3) were soaked in 1 � Tris-buffered saline (TBS) solutionfor 10 min. Later, the filter papers and membrane were arranged inthe apparatus and connected to a vacuum pump (Millipore). Themembranes were rehydrated by using 1 � TBS (100 mL) by vacuumfiltration. Subsequently, the samples (50 mL) were blotted on themembranes and washed with 1 � TBS (200 mL) through application

of vacuum. The blot was blocked with 5 % blotto for 1 h at RT. Im-munoblot analysis was performed as described previously by usingUVP, biospectrum 810 imaging system.[27]

Tubulin polymerization assay : An in vitro assay for monitoring thetime-dependent polymerization of tubulin to microtubules wasperformed by employing a fluorescence-based tubulin polymeri-zation assay kit (BK011, Cytoskeleton, Inc.) according to the manu-facturer’s protocol. The reaction mixture in a final volume of 10 mLin PEM buffer [80 mm piperazine-N,N’-bis(2-ethanesulfonic acid)(PIPES), 0.5 mm ethylene glycol tetraacetic acid (EGTA), 2 mm MgCl2,pH 6.9] in 384-well plates contained bovine brain tubulin(2 mg mL�1), 10 mm fluorescent reporter, 1 mm guanosine-5’-tri-phosphate (GTP) in the presence or absence of test compounds at37 8C. Tubulin polymerization was followed by monitoring the fluo-rescence enhancement resulting from the incorporation of a fluo-rescence reporter into the microtubules as the polymerization pro-ceeded. Fluorescence emission at l= 420 nm (lexc = 360 nm) wasmeasured for 1 h at 1 min intervals in a multimode plate reader(Tecan M200). To determine the IC50 values of the compoundsagainst tubulin polymerization, the compounds were preincubatedwith tubulin at varying concentrations (0.01, 0.1, 1, 10, and100 mm). Assays were performed under conditions similar to thoseemployed for polymerization assays as described above.[29]

Analysis of cell cycle : A549 cells in 60 mm dishes were incubatedfor 24 h in the presence or absence of test compounds 3 e, 3 g,and 4 e at 1 mm concentration. Cells were harvested with trypsin–EDTA and fixed with ice-cold 70 % ethanol at 4 8C for 30 min; then,ethanol was removed by centrifugation, and the cells were stainedwith DNA staining solution [1 mL; 0.2 mg of propidium iodide (PI),and 2 mg RNase A] for 30 min as described earlier. The DNA con-tents of 20 000 events were measured by flow cytometer (BD FACS-Canto II). Histograms were analyzed by using FCS express 4 plus.

Immunohistochemistry of tubulin and analysis of nuclear morpholo-gy : A549 cells were seeded on glass cover slip and incubated for24 h in the presence or absence of test compounds at a concentra-tion of 1 mm. Cells grown on coverslips were fixed in 3.5 % formal-dehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 min at RT.Cells were permeabilized for 6 min in PBS containing 0.5 % TritonX-100 (Sigma) and 0.05 % Tween-20 (Sigma). The permeabilizedcells were blocked with 2 % bovine serum albumin (Sigma) in PBSfor 1 h. Later, the cells were incubated with the primary antibodyfor tubulin (Sigma) at 1:200 diluted in blocking solution for 4 h atRT. Subsequently, the antibodies were removed, and the cells werewashed with PBS (3 �). Cells were then incubated with fluoresceinisothiocyanate labeled anti-mouse secondary antibody (1:500) for1 h at RT. Cells were washed with PBS (3 �) and mounted inmedium containing 4’,6-diamidino-2-phenylindole. Images werecaptured by using the Olympus confocal microscope FLOW VIEWFV 1000 series and analyzed with FV10ASW 1.7 series software.

Western blot analysis of soluble versus polymerized tubulin : Cellswere seeded in 12-well plates at 1 � 105 cells per well in completegrowth medium. Following individual treatment of cells with com-pounds 3 e, 3 g, and 4 e for 24 h, the cells were washed with PBSand subsequently soluble and insoluble tubulin fractions were col-lected. To collect the soluble tubulin fractions, cells were permeabi-lized with prewarmed lysis buffer [200 mL; 80 mm PIPES-KOH(pH 6.8), 1 mm MgCl2, 1 mm EGTA, 0.2 % Triton X-100, 10 % glycerol,0.1 % protease inhibitor cocktail (Sigma–Aldrich)] and incubated for3 min at 30 8C. The lysis buffer was gently removed and mixedwith 3 � Laemmli’s sample buffer [100 mL; 180 mm Tris-Cl pH 6.8,6 % sodium dodecyl sulfate (SDS), 15 % glycerol, 7.5 % b-mercap-




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toethanol, 0.01 % bromophenol blue]. Samples were immediatelyheated to 95 8C for 3 min. To collect the insoluble tubulin fraction,1 � Laemmli’s sample buffer (300 mL) was added to the remainingcells in each well, and the samples were heated to 95 8C for 3 min.Equal volumes of samples were run on an SDS–10 % polyacryl-amide gel and were transferred to a nitrocellulose membrane em-ploying semidry transfer at 50 mA for 1 h. Blots were probed withmouse anti-human a-tubulin diluted 1:2000 mL (Sigma) andstained with rabbit anti-mouse secondary antibody coupled withhorseradish peroxidase, diluted 1:5000 mL (Sigma). Bands were vi-sualized by using an enhanced chemiluminescence protocol(Pierce) and radiographic film (Kodak).[31]

Molecular modeling

Optimizations of all compounds were performed in Gaussian 09 byusing PM3 semiempirical methods. The protein cocrystal structurewas downloaded from RSCB-Protein Data Bank with ligand colchi-cine (PDB ID 3E22). [32] Autodock 4.2 software-[33] was used to per-form the docking studies. The visualization and analysis of the in-teractions were performed by using PyMOL (ver. 0.99). [34]

Acknowledgements

A.B.S. , G.B.K. , V.S.R. , M.K.R. , and C.R.R. acknowledge CSIR-UGC,New Delhi, for the award of senior research fellowships. The au-thors also acknowledge the Council of Scientific and IndustrialResearch (CSIR), India for financial support under the 12th FiveYear plan project “Affordable Cancer Therapeutics (ACT)”(CSC0301) and “Small Molecules in Lead Exploration (SMiLE)”(CSC0111).

Keywords: antiproliferation · cell-cycle arrest · dot-blotanalyses · molecular docking · polymerization

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FULL PAPERS

A. Kamal,* G. B. Kumar, S. Polepalli,A. B. Shaik, V. S. Reddy, M. K. Reddy,C. R. Reddy, R. Mahesh, J. S. Kapure,N. Jain

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Design and Synthesis ofAminostilbene–Arylpropenones asTubulin Polymerization Inhibitors

Sittin’ on the docking site of tubulin:A series of aminostilbene–arylprope-nones are designed, synthesized, andevaluated for their cytotoxic activity.These compounds inhibit microtubuleassembly and induce cyclin B1 proteinexpression. Docking experiments revealthat these compounds bind with thecolchicine binding site of tubulin.



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Design and Synthesis of Aminostilbene-Arylpropenones as Tubulin Polymerization Inhibitors

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